Functional Identification Of Millet Transcription Factor Gene SibZIP42 And SiNAC18 Involved In Abiotic Stress Tolerance

Abiotic stress such as drought and salinity seriously affected the yield and quality of crops,foxtail millet(Setaria italica L.)characteristic with drought resistance,barren tolerance,strong adaptability,is an ideal material for studying monocotyledonous crops abiotic stress response.bZIP(the basic leucine zipper)and NAC(nascent polypeptide-associated complex)transcription factors play important roles in plant growth and development as well as the abiotic stress response process.In this study,we selected bZIP transcription factors SibZIP42 and NAC transcription factor SiNAC18 which expression level up-regulated 2 times from millet drought transcriptome sequencing,using sequence alignment,characterization analysis,gene functional identification and downstream gene detection methods to sesearch the molecular mechanism of SibZIP42 and SiNAC18 improve the plant resistance to salt and drought resistance.In this research,the material Longgu 25’s cDNA is as template,to complete SibZIP42 and SiNAC18 transcription factor gene amplification,respectively.The functional of these two transcription factor genes were characterized,and get the following results:1.The results of millet transcription factor gene SibZIP42(1)Phylogenetic tree analysis showed that SibZIP42 located in bZIP family S subgroup,with the highest homology 89% with Arabidopsis thaliana gene Atb ZIP42;promoter element analysis showed that SibZIP42 contains ABRE,MYB and MYC components in response to a variety of stress;phosphorylation site analysis showed that SibZIP42 contains several phosphorylation sites.(2)Real-Time PCR results showed that SibZIP42 response to various abiotic stresses in varying degrees,under the treatment of salnity,drought and ABA,the realetive expression level was significantly increased;The analysis of tissue specific expression showed that SibZIP42 expression in root was significantly higher than that in stems and leaves;The result of subcellular localization indicated that SibZIP42 protein was localized in the nucleus.(3)Gene function analysis results show that the transgenic Arabidopsis germination rateand greening rate was higher than that of the WT when MS medium contains NaCl.(4)The transgenic Arabidopsis plants greening rate was significantly lower than that of WT when MS medium contains ABA;The downstream gene detection results showed that ABA stress response related genes(HIS1-3,RD29 B and RAB18)and the response to dehydration related gene(PIP2A)in transgenic plants was significantly higher than the expression in WT.The results showed that SibZIP42 could improve plant tolerance to high salt stress through ABA signaling pathway.2.The results of millet transcription factor gene SiNAC18(1)The full-length of SiNAC18 is 1074 bp,encoding a hydrophilic protein consists of 357 amino acid,the molecular weight is about 38.8KD,and isoelectric point was 6.33,multiple sequence alignment analysis showed that SiNAC18 contains 5 NAC conservative structure domain contains A,B,C,D,E;Phosphorylation site analysis showed that SiNAC18 protein contains several phosphorylation sites;The results of subcellular localization showed that SiNAC18 protein localized in nucleus.(2)The expression pattern analysis showed that the expression of SiNAC18 was significantly increased under drought and salt stress.(3)The results of gene function analysis showed that under PEG treatment,the germination of SiNAC18 transgenic Arabidopsis was higher than that of WT.(4)The transgenic lines increased the sensitivity to ABA;The downstream gene detection results show that the expression level of ABA stress response related genes(P5CR,PIP2 A,POD,RD29A)in SiNAC18 over expression lines was significantly higher than that of the WT,suggesting that SiNAC18 improve drought tolerance in plant by enhancing the expression of these related genes.